Method for generating t cells progenitors

ABSTRACT

The invention relates to an in vitro method to generate T cell progenitors, comprising the step of culturing CD34+ cells in a medium containing TNF-alpha and/or an antagonist of the Aryl hydro-carbon/Dioxin receptor, in particular StemRegenin 1 (SR1), in presence of a Notch ligand and optionally a fibronectin fragment.

The invention relates to the field of cell therapy, in particular ofhematopoietic stem cells graft, transformed or not, and moreparticularly of immune reconstitution after such graft.

Graft of progenitor and Hematopoietic Stem/Progenitor Cell (HSPC) isconsidered the best therapeutic option for the most severe hereditaryimmune deficiencies, for many malignant hemopathies, as well as for anumber of solid tumors.

Currently, in allograft situations with partial HLA incompatibility, theinjections, to previously conditioned recipients, of increasing doses ofsorted CD34+ HSPC allows donor transplantation with effective preventionof graft-versus-host disease (GVH). Nevertheless, the differentiation ofnew T lymphocytes from the injected CD34+ cells requires a minimumperiod of 4 months and these T lymphocytes are in sufficient number toplay a protective role against infections only a few months after theirappearance.

This slowness of immune reconstitution leads to numerous infectiouscomplications especially viral, but also to relapses, which influencethe long-term prognosis of the grafted patients.

In addition, other therapeutic protocols use a gene therapy approach,namely an autograft of transduced HSPC, which has been shown to beeffective in the treatment of certain hereditary immune deficiencies.The advantage of this strategy over HSPC CD34+ allogeneictransplantation is indisputable in terms of survival and morbidity whenno HLA-compatible donor is available. Nevertheless, clinical experiencehas shown that, for some patients with severe infections, reconstitutionof the T lymphocyte compartment is slow and never reaches normal levelsof circulating T lymphocytes. The morbidity and mortality associatedwith this particular context are important.

Because of the high morbidity and mortality associated with this type oftransplant, the development of novel therapies to reduce theimmunodeficiency period after transplantation is fully justified.

In particular, it is important to accelerate the generation of Tlymphocytes by the administration of precursors already engaged in the Tlymphocyte differentiation pathway (T cell progenitors).

These T cell precursors are obtained from CD34+ HSPC differentiation andhave in particular the CD7+ marker, which is a marker of differentiationin the T-cell pathway. They may also have other markers. Awong et al(Blood 2009; 114: 972-982) described the following precursors of Tlymphocytes: early thymic progenitor (ETP), which have markers(CD34+/CD45RA+/CD7+), precursor cells at the proT1 stage (CD7++/CD5−),precursor cells at the proT2 stage (CD7++/CD5+), and cells at the preTstage (CD7++/CD5+CD1a+). The HSPC acquire these markers in a successiveway when passing from one stage to the other, over the T celldevelopment pathway. It is further known that in humans, the CD1aantigen distinguishes the passage from a very immature thymic progenitorto a progenitor clearly engaged in the T-pathway (Cavazzana-Calvo et al,MEDECINE/SCIENCES 2006; 22: 151-9).

A T-cell precursors transplant, concomitant with HSPC grafting, wouldallow the rapid production of a mature and functional T lymphocytecompartment and thus help prevent the risk of severe infections byallowing the patient to benefit from some immunity before completereconstitution of the immune system.

Moreover, it is important to be able to use adult cells rather than cordblood cells, since it is easier and cheaper to obtain adult cells thancord blood cells and since adult cells are more commonly used inallograft.

However, data published in the literature, obtained in humans and mice,show intrinsic differences between fetal hematopoietic cells (includingcord blood) and adult cells. These differences relate to survival, theability to repair DNA damage, proliferative capacity and potential todifferentiate (see, for example, Yuan et al., (2012 Mar. 9; 335 (6073):1195-200), which Indicate that adult bone marrow cells are lesseffective than fetal cells in their potential to generate a variety ofcell types: Lansdorp et al (J Exp Med 1993 Sep. 1; 178 (3): 787-91);Szilvassy et al (Blood, 2001 Oct. 1, 98 (7): 2108-15), Frassoni et al(Blood, 2003 Aug. 1, 102 (3): 1138-41), Liang et al. 106 (4): 1479-87),Six et al (J Exp Med 2007 Dec. 24, 204 (13): 3085-93)).

WO 2016/055396 describes that it is possible to generate T-cellprecursors by culturing CD34+ cells in presence of an immobilized ligandof Notch (in particular the soluble domain of the Delta-like-ligand,fused to a Fc region of an IgG protein), and of a fragment of afibronectin, containing the RGDS (SEQ ID NO: 3,Arginine-Glycine-Aspartate-Sérine) and CS-1 domains, as well as theheparin-binding domain (in particular in the presence of Retronectin®).The Notch ligand used may be referred to as DL4/Fc.

It is to be noted that this document discloses that presence of both theimmobilized ligand of Notch, and the fibronectin makes it possible toincrease generation of T lymphocytes progenitors (CD7+ cells), which wasalready an improvement in the art, but that the percentage of CD7+CD34−cells remains quite low, as shown in FIG. 3 of WO 2016/055396. It ishowever interesting and particularly important to increase thispercentage, in order to be able to administer a higher amount ofprecursor to the patient in need thereof. On the other hand, it is alsoimportant that the cells remains at a early stage of differentiation tobe able to provide a proper immunity.

It is reminded that Notch proteins are transmembrane receptors thatregulate the cellular response to a large number of environmentalsignals. In mammals, four Notch (Notch 1-4) receptors and five ligands(Delta-like-1, 3, and 4, Jagged1, Jagged2) have been described(Weinmaster Curr Opin Genet Dev 2000: 10: 363-369).

The Delta-like-ligand 4 can be designated as:

-   -   (ii) Delta-like-ligand 4 (corresponding to the name of the DLL4        gene)    -   (iii) Delta-like-4 or Delta ligand 4 (abbreviation DL-4).

In the present application, the Notch Delta-like-1 and Delta-like-4ligands may be designated respectively by DL1 and DL4 or by DL-1 andDL-4. The sequences of ligands DL-1 and DL-4 are specified as SEQ ID NO:1 and SEQ ID NO: 2, respectively.

Ohishi et al (BLOOD, vol. 98, no. 5, 2001, pp 1402-1407) relates to theeffect of Notch signaling on monocyte differentiation into macrophagesand dendritic cells. The monocyte cells used in the experimentsdisclosed in this document were either peripheral blood monocytespurified by negative selection, or monocytes obtained after in vitrodifferentiation of CD34+ stem cells. The cells used in this documenthave thus entered the monocyte/macrophage/dendritic cellsdifferentiation pathway, have lost the CD34 marker (which is ahematopoietic stem cell marker) and present the CD14 marker. These cellsare cultured in presence of the extracellular domain of Delta-1 andGM-CSF, TNF-alpha (which is used to induce the differentiation of CD34+cell-derived CD1a-CD14+ cells into dendritic cells). This document thisdoesn't uses CD34+ cells in presence of a Notch ligand and of TNF-alphaand doesn't pertain to the generation of T-cell precursors (CD7+Tlymphocytes precursors).

SHUKLA et al (NATURE METHODS, vol. 14, no. 5, 2017, 531-538) and WO2017/173551 disclose a method for generating progenitor T cells fromstem and/or progenitor cells comprising exposing the stem and/orprogenitor cells to Notch ligand Delta-like-4 (DL4) and vascularadhesion molecule 1 (VCAM-1).

The method described in the present application allows the generationand the increase (proliferation) of number of CD7+T lymphocyteprecursors, from CD34+ stem cells, without using a cell stroma (whichcan't be easily envisaged in a clinical context). Furthermore, themethod is particularly adapted when performed with CD34+ cells issuedfrom adult donors.

Furthermore, the cells obtained through the process as herein disclosed,harbor the BcI11b marker, which is important transcriptional factoruniquely switched on since T-cell commitment (Kueh et al, 2016, NatImmunol. 2016 August; 17(8):956-65. doi: 10.1038/ni.3514).

The inventors have also shown absence of rearrangement of the T cellreceptors loci (either TCRbeta, TCRgamma or TCRdelta) in the progenitorsobtained through the process herein disclosed.

Moreover, a decrease in apoptosis markers was shown, for the precursorsherein obtained, as compared to the process disclosed in WO 2016/055396.

In summary, the process herein disclosed makes it possible to obtain anumber of T cell precursors high enough to be efficiently used for anadult receiving a stem cells transplant.

This method may also be used to obtain transformed (transduced) T cellprecursors for gene therapy, when a vector containing a gene of interestis used at some point during the process herein disclosed.

The cells obtained by the process herein disclosed can be obtained foradult CD34+ cells, and can be used for allogenic grafts or autografts,even when cord blood cells are used for such grafts.

It is to be noted that the method, although very efficient for adultCD34+ cells, may also be used with cord blood CD34+ cells.

In a particular embodiment, the method herein disclosed thus fastenT-cell generation in vivo after injection of T-cell progenitors producedfrom HSPC in an in vitro culture system, combining an immobilized fusionprotein derived from the Notch ligand, Delta-4, Retronectin® and acombination of cytokines.

This system allows, within 7 days, the generation of T-cell progenitorsthat are phenotypically and molecularly similar to human thymic T-cellprecursors. Furthermore, these T-cell progenitors are able to give risehuman mature and diverse T-cell in NSG mice, with a faster kinetic ascompared to HSPC.

The results herein reported were obtained from both cord blood (CB) andadult (mobilized peripheral blood, mPB from adult donors) HSPC.

The inventors have shown that the amount of T cell precursors can beimproved from CD34+ cells by exposing said CD34+ cells to a Notch ligandand in the presence of the soluble TNF-alpha (Tumor Necrosis FactorAlpha, Uniprot P01375, RefSeq NP_000585, SEQ ID NO: 8). Said exposure ismade under conditions suitable to generate progenitor T cells.Optionally, the cells are also exposed to a fibronectin fragmentcontaining an RDGS motif, and/or a CS-1 motif and optionally a heparinbinding domain. Preferably, said fibronectin fragment contains an RDGSmotif, a CS-1 motif and a heparin binding domain.

TNF is primarily produced as a 233-amino acid-long type II transmembraneprotein arranged in stable homotrimers. The secreted form of humanTNF-alpha takes on a triangular pyramid shape, and weighs around 17-kDa.

As disclosed in WO 2016/055396, it is possible to perform the methodherein disclosed, using a RGDS peptide and/or a CS-1 peptide in place ofthe fibronectin fragment. A combined use of the RGDS and CS-1 peptidesis preferred, in particular fused in the same protein. Thus, the RGDSand/or CS-1 peptides may be present as such in the culture medium orwithin a polypeptide or protein present in the culture medium. When theculture medium only contains the RGDS and/or CS-1 peptide as such, onecan relate to these as “free” peptide(s) in the culture medium if suchpeptides are not immobilized on the inner surface of the culture vessel.Indeed, the peptide(s) may be in solution or immobilized on the innersurface of the vessel in which the CD34+ cells are exposed to theimmobilized Notch ligand.

However, as indicated above, it is preferred to use a fragment offibronectin, which contains the RGDS and CS-1 patterns, as well as aheparin-binding domain. The fibronectin fragment may be free in solutionor immobilized on the inner surface of the culture container.

The process is performed in vitro, in a container, such as a cellculture plate (Petri dish, 24 well array or the like), preferably withthe Notch ligand immobilized on its inner surface. The Notch ligand may,however, be immobilized on any other support present in the culturemedium, such as on the surface of beads (in particular microbeads).Immobilization of the Notch ligand is essentially intended to stabilizethe ligand, in order to allow activation of the Notch receptor of theCD34+ cells.

By “T cell progenitor”, one intends to designate any cell involved inthe differentiation pathway to the T lymphoid pathway from a CD34+ HSPC.This cell is therefore characterized in that it expresses the CD7marker, which is known to be one of the earliest markers during thelymphopoiesis of the T cells. Depending on the state of differentiationin the T lymphoid pathway, it can express or not the CD34 marker (lossof CD34 during differentiation). Such T cell progenitor may also expressor not the CD5 marker.

Among the “T-cell progenitors” are those cells which can be found in thepost-natal thymus, i.e. early thymic progenitor (ETP)(CD34+/CD45RA+/CD7+), proT1 cells (CD34+CD45RA+CD7++CD5−CD1a−), proT2cells (CD34+CD45RA+CD7++CD5+CD1a−) and preT cells(CD34−CD7++/CD5+CD1a+). T-cell receptor (TCR) loci rearrange in a highlyordered way (TCRδ-TCRγ-TCRβ-TCRα). To note, the first functional TCRrearrangements occurs at the CD34-CD7++/CD5+CD1a+ preT cell stage (Diket al. J Exp Med 2005; 201:1715-1723). T-cell progenitors are well knownin the state of the art. They are cited in particular by Reimann et al(STEM CELLS 2012; 30:1771-1780.) and by Awong et al (2009, op.cit.).

The term “RGDS peptide” is intended to designate any peptide or proteinthat contains the RGDS pattern, so that it can bind integrin VLA-5. Suchpeptide or protein can be tested for its ability to bind VLA-5 integrinby methods known and reported in the art. RGDS peptide binds to integrinVLA-5 (Very Late Antigen-5), which is a dimer composed of CD49e (alpha5)and CD29 (beta1).

Heparin-binding domains are known in the art and present in numerousproteins that bind to heparin. Their sequence is generally XBBXBX orXBBBXXBX (B=acide aminé basique; X=acide aminé hydropathique; Cardin andWeintraub, Arterioscler Thromb Vasc Biol. 1989; 9:21-32, SEQ ID NO: 4 etSEQ ID NO: 5).

Presence of such a heparin-binding domain is particularly favorable whenthe CD34+ cells are exposed to a viral (especially a retroviral) vectorin order to transduce them and obtain T cell progenitors expressing atransgene.

A CS-1 peptide or CS-1 pattern is a 25 amino acids peptide(DELPQLVTLPHPNLHGPEILDVPST, SEQ ID NO: 6), described by Wayner et al,1989, J. Cell Biol. 109: 1321). This CS-1 pattern binds to the VLA-4(Very Late Antigen-4) receptor. This antigen is a dimer integrin,composed of CD49d (alpha 4) and CD29 (beta 1).

In a particular embodiment, the fibronectin fragment is present in theculture medium or immobilized on the inner (in particular the bottom)wall of the container. Fibronectin is a protein, which in its naturalform is a v-shaped large dimer of 100 nm long and 460 kDa. The twomonomers are connected by two disulfide bridges at their C-terminus. Theterm “fibronectin” or “fibronectin fragment” is understood to mean thenatural fibronectin protein (i.e. any isoform produced by alternativesplicing), but also a monomer of this protein, or a fragment of thisprotein (but containing the peptide RGDS, as well as CS-1 peptide andheparin binding site).

A fibronectin which is particularly suitable for carrying out theprocess herein disclosed is Retronectin®. This protein corresponds to afragment of a human fibronectin (CH-296 fragment, Kimizuka et al., JBiochem., 1991 August 110 (2):284-91, Chono et al., J Biochem 2001September 130 (3):331-4) and contains the three functional domains thatare preferred for implementation of the method (the cell-binding Cdomain containing the RGDS peptide, the heparin-binding domain and theCS-1 sequence). This protein is sold in particular by the company TakaraBio Inc. (Shiga, Japan).

In a particular embodiment, the fibronectin fragment is immobilized(i.e. bound to a solid support and is not present free in solution(although it is possible that certain elements may be found insolution)). This solid support is preferably the bottom wall of thecontainer in which the process is carried out. However, it is alsopossible to envisage binding the fibronectin fragment to beads, such aspolymer or magnetic beads (with a diameter generally comprised between 1and 5 μm). The binding of the protein or peptide to these beads may ormay not be covalent. Methods for attaching a protein or peptide to thebeads are known in the art. It is also possible to introduce thefibronectin fragment into a semisolid medium, such as agar or gel.

When the fibronectin fragment is immobilized on the support (inparticular the bottom wall of the container in which the process isperformed), this immobilization may also be covalent or not. In apreferred embodiment, this immobilization is carried out non-covalentlyby allowing the fibronectin fragment to be absorbed onto the glass orplastic composing the bottom wall of the container.

In a particular embodiment, as seen above, the differentiation of theCD34+ cells into T-cell progenitors is carried out together with thetransduction or transfection (including Nucleofection™, a specificelectroporation system developed by Lonza) of the CD34+ cells by meansof a vector (such as a viral vector or a nucleic acid fragment such as aplasmid or plasmidic RNA or DNA sequences) in order to introduce a geneof interest (or a system for gene editing) in these cells. This meansthat the cells exposed to the Notch ligand and the fibronectin fragment,with TNF-alpha, are also exposed to a viral supernatant for at leastpart of their time of exposure to the Notch ligand and fibronectinfragment, with TNF-alpha.

The teachings of WO 2016/055396 with regards to the operative conditionsfor performing cell transduction are expressly applicable to the presentmethod and are thus considered as being expressly recited in thisapplication.

One can cite, in particular:

-   -   (iv) Exposure of the cells to the Notch ligand, fibronectine        fragment and TNF-alpha, for some time (preferably more than 4        hours, more preferably more than 6 h, or more than 8 h or 10 h,        but less than 36 h, more preferably less than 30 h or less than        24 h) with appropriate cytokines known in the art and disclosed        in WO 2016/055396, and addition of the viral supernatant for an        appropriate duration (preferably more than 4 hours, more        preferably more than 6 h, 8 h or 10 h, and preferably less than        30 hours, more preferably less than 24 h, more preferably around        16 h).    -   (v) Second transduction if required as taught in WO 2016/055396    -   (vi) Use of this protocol for autologous hematopoietic stem        cells grafts in gene therapy protocols, in order for the        transgene to add a protein that is absent or deficient in the        patient so as to bring a therapeutic benefit.    -   (vii) Usable transgenes: gene correcting immunodeficiencies (in        particular severe combined immunodeficiencies SCID or not, CID),        HIV, X-linked adrenoleukodystrophy, hemoglobinopathies, in        particular β-thalassemy or sickle cell disease. One can also        use, as the transgene, a gene that codes for a Chimeric Antigen        Receptor (CAR), i.e. a cell surface protein that recognizes a        cell surface protein that is specifically expressed by cancer        cells in order to trigger immune response against the cancer        cells through the engineered CAR-T cells    -   (viii) Preferred use of a viral supernatant to allow insertion        of the transgene within the cell genome, using in particular        lentiviruses known, and described in the art.    -   (ix) Introduction of the viral vector in the cell culture medium        after pre-activation of the CD34+ cells between 4 and 36 h,        preferably between 6 and 24 h    -   (x) Exposure of the cells to the viral vector between 4 and 30        h, preferably between 12 and 24 h, more preferably around 16 h,        and removal of the viral vector (harvesting and washing the        cells, and resuspending these cells in presence of the Notch        ligand, the fibronectin fragment and the TNF-alpha).

As indicated above, in another embodiment, the CD34+ cells are exposedto a system making it possible to perform gene editing. Such systems arenow widely known and described in the art and are essentially based onnucleic acid double-break repair. Such genome editing system may use anuclease selected from the group consisting of meganucleases, zincfinger nucleases (ZFNs), transcription activator-like effector-basednucleases (TALEN), and CRISPR-Cas nucleases. The fact that the CD34+cells proliferate during exposure to the elements as recited above, andthus in presence of TNF-alpha, makes it possible to use these geneediting systems that require proliferation of cells.

In a particular embodiment, the Notch ligand is the Delta-like-1 protein(SEQ ID NO: 1) or a fragment thereof (soluble domain).

In another and preferred embodiment, the Notch ligand is theDelta-like-4 protein (SEQ ID NO: 2).

In a particular embodiment, said Notch ligand is a fusion proteincomprising the soluble domain of a natural Notch ligand fused to an Fcregion of an IgG protein. As known in the art, the soluble domain of aNotch ligand represents the extracellular portion of said ligand.Varnum-Finney et al (J Cell Sci., 2000 December; 113 Pt 23:4313-8)described a fusion protein of the soluble part of DL-1 with an Fcportion of IGg1. Reimann et al (op cit) described a fusion protein ofthe soluble part of DL-4 (amino acids 1-526) with the Fc fragment of anIgG2b immunoglobulin. It is thus preferred when the IgG protein is anIgG2. In a preferred embodiment, the sequence of the Notch ligand usedin the method herein disclosed is SEQ ID NO: 7. A commercially availableproduct (Sino Biologicals) comprising the extracellular domain (Met1-Pro 524) of human DLL4 (full-length DLL4 accession number NP_061947.1)fused to the Fc region of human IgG 1 at the C-terminus is a DL4 proteinis also suitable for use herein.

The culture medium used in the context of the method herein disclosed isany medium adapted for culturing CD34+ cells and T cells. Mention may inparticular be made of α-MEM, DMEM, RPMI 1640, IMDM, BME, McCoy's 5A,StemSpan™, in particular SFII (StemCell Technologies) media or Fischer'smedium. StemSpan™ SFII medium contains, in particular, Iscove's MDM,Bovine serum albumin, Recombinant human insulin, Human transferrin(iron-saturated), 2-Mercaptoethanol.

A suitable and preferred culture medium for carrying out the processherein disclosed is the X-VIVO™ medium (Lonza, Basel, Switzerland). Thismedium was used in particular by Jonuleit et al (Eur J Immunol, 1997,27, 12, 3135-42) and Luft et al (J Immunol, 1998, 161, 4, 1947-53).

Preferably, a basal medium is used (i.e., which a medium that allows thegrowth of the cells without the need to add supplements), in which,however, one preferably would add serum, and/or growth factors andcytokines.

Thus, fetal bovine serum (FBS) or fetal calf serum (FCS), autologoushuman serum or human AB serum is preferably added to the basal culturemedium. Preferably, this medium is supplemented with at least 15% offetal serum, more preferably at least 20%. The FBS is particularlysuitable for the implementation of the process. In particular, it ispreferred to use defined FBS. The defined FBS is a high-quality serumwhich has been analyzed and filtered to avoid the presence of viralparticles. It is sold as such by many suppliers, such as the HyClone™Defined Fetal Bovine Serum (FBS) of Thermo Scientific™.

In addition to TNF-alpha, the culture medium is also preferablycomplemented with cytokines and growth factors. These cytokines andgrowth factors are especially selected from the group consisting of SCF(stem cell factor), thrombopoietin (TPO, also called megakaryocytegrowth and development factor, MGDF), Flt3-Ligand (which is a growthfactor Hematopoietic), interleukin 3 (IL-3), interleukin 7 (IL-7) andSCF (stem cell factor). In a particular embodiment, the culture mediumcontains at least three, preferably at least four of these cytokines orgrowth factors, in addition to TNF-alpha.

In a preferred embodiment, and in particular for the generation of Tcell precursors that are not transduced with a viral vector, at least orexactly three cytokines are added. Preferably, these three cytokines areInterleukin-7 (IL-7), SCF (Stem Cell Factor) and Flt-3 Ligand(hematopoietic growth factor).

In another preferred embodiment, four cytokines, i.e. the threecytokines mentioned above and TPO (thrombopoietin) are added.

In another particular embodiment, and in particular for the generationof T cell precursors transduced with a viral vector, the nature of thecytokines and growth factors can be varied during the implementation ofthe method.

Thus, IL-3, IL-7, SCF, TPO, and Flt3-L can be used in the medium if thestep of pre-activating the cells prior to addition of the viral vector,and then supplement the medium only with IL-7, SCF, TPO, and Flt3-Lafter the vector is removed.

The aforementioned cytokine and growth factor mixtures are sufficient toinduce the differentiation of CD34+ cells into T-cell precursors andgenerally the culture medium comprises no other cytokine or growthfactor, except TNF-alpha, which, as described in the examples, makes itpossible to increase the number of T cell precursors.

In the process herein foreseen, the total duration of exposure of theCD34+ cells in the presence of the Notch ligand, of the protein orpeptide exhibiting the RGDS motif and of TNF-alpha, is generally carriedout for a time preferably of more than 3 days and less than 10 days.

This exposure may vary depending on whether the cells are transduced.Thus, an exposure time of three days may be sufficient for adult stemcells not transduced, whereas it will generally be longer for infantilestem cells (about 7 days) or when transduction is performed.

CD34+ cells are obtained from a bone marrow puncture, from umbilicalcord blood or from peripheral blood from adult donors, which have beenmobilized particularly with G-CSF or any other mobilizing agent known inthe art. Methods for sorting CD34+ cells are known in the art. Inparticular, magnetic beads having an antibody recognizing CD34 on theirsurface can be used for this purpose.

Preferably, the cell culture vessel is prepared by immobilizing theNotch ligand and the fibronectin fragment on the inner (preferably thelower) surface prior to exposing the CD34+ cells. Retronectin® or otherfibronectin naturally adhere to the plastic of the cell culture box(Petri dish or 24-well box, or other). Similarly, if Notch ligand isused as a fusion protein with the Fc fragment of an immunoglobulin, thisFc fragment also adheres to the plastics. It is therefore sufficient toleave the container in the presence of these compounds for a few hoursin order to obtain the appropriate coating. A method to coat suchculture container with the Notch ligand and the fibronectin fragment isdisclosed in details in WO 2016/055396 and can be applied forimplementing the method as herein disclosed.

It is reminded that, according to WO 2016/055396, around 75% of DL-4will adhere to the container surface when 5 μg/ml is used, the optimaldose being higher or equal to 1.25 μg/ml and preferably between 2.5 and5 μg/ml.

With regards to the fibronectin fragment, a concentration of 25 μg/ml isparticularly adapted (especially when Retronectin® is used), althoughone can use other concentrations (higher or lower).

In the implementation of the process herein disclosed, CD34+ cells areadded at a concentration comprised between 10⁶ et 10⁷ cells/ml, inparticular around 2×10⁶ CD34+ cells/ml in the culture vessel.

Depending on whether the cells are to be transduced and of the CD34+cells, one can use a plate of between 2 to 10 cm² (i.e. a plate from 24to 6 wells). When one uses a 24 wells plate, one adds between 10⁴ and10⁶ CD34+ cells in each well, preferably from 2×10⁴ to 4×10⁵ CD34+ cellsper well. When a 6 well plate is used, one will add between 8×10⁴ and2×10⁶ cells per well.

The quantity of cells to add is adapted by the person skilled in theart, according to the container used.

The cells are placed in the well, in the selected basal medium,supplemented with TNF-alpha, and preferentially supplemented with growthfactors and cytokines, as indicated above.

The concentrations of cytokines or growth factors are between 2 and 300ng/ml. Preferably, the concentration is higher than 40 ng/I and lowerthan 300 ng/ml or 200 ng/I, more preferably around 100 ng/ml.

However, when one wishes to generate transduced T cell progenitors, andwhen the cells are pre-activated before being exposed to the viralvector, one can use higher concentrations (around 300-400 ng/ml). Inthis embodiment, SCF and Flt3-L can be used at des concentrations in therange of 300 ng/ml, TPO and IL-7 in the range of 100 ng/ml, and IL-3 atabout 40 ng/ml.

TNF-alpha is preferably used at a concentration equal or higher than 5ng/ml. Indeed, as demonstrated in the examples, this low concentrationis enough to obtain the proliferation of the T cell precursors, withoutmodifying the differentiation pathway.

However, higher concentrations can also be used. In particular, theTNF-alpha concentration can be as high as 300 ng/ml or 200 ng/ml. Anappropriate concentration is around 100 ng/ml. However, otherconcentrations such as 10 ng/ml, 20 ng/ml or 50 ng/ml are also suitable.

The invention thus relates to an in vitro method for generating T cellsprecursors comprising the step of culturing CD34+ cells in a suitablemedium comprising TNF-alpha and in the presence of an immobilized Notchligand, and optionally the fibronectin fragment as disclosed above.

The invention thus relates to an in vitro method for expanding T cellsprecursors comprising the step of culturing CD34+ cells in a suitablemedium comprising TNF-alpha and in the presence of an immobilized Notchligand, and optionally the fibronectin fragment as disclosed above.

Expanding the T cells precursors is intended to mean that there are moreT cell precursors than when the TNF-alpha is not used, and/or that thereare more T cell precursors than the number of CD34+ cells introduced inthe container.

In particular, the T cells precursors obtained by the above methods areCD34−/CD7+/CD5− precursors.

Generally, the cells don't harbor the CD1a marker either.

In a preferred embodiment, the T cells precursors obtained by the abovemethods are CD34−/CD7+/CD1a− precursors.

This means that the population of T cell precursors obtained by thepresent method express the CD7 marker, and that more than 80% of thecells expressing this marker have the above phenotype (not expressing

-   -   CD34 and CD1a, or    -   CD34 and CD5, or    -   CD34 and CD1a and CD5),

as analyzed by flow cytometry. This phenotype is generally obtainedafter 7 days of culture.

As indicated, the CD34+ cells are preferably not cord blood cells.However, the method may also be used with and is applicable to cordblood CD34+ cells, and it also leads to improved results.

The method is particularly interesting when implemented with CD34+ cellsthat have been isolated from an adult patient. Said patient may be ahealthy donor, or a donor with a disease, and particularly for which thecells will be corrected by viral transduction.

The cells may be cultured for more than 3 days, and preferably for atleast 5 days, more preferably for at least or exactly 6 days, mostpreferably for at least or exactly 7 days, although the duration ofculture may last longer.

In the method herein disclosed, TNF-alpha is preferably added to theculture medium from day 0 (i.e. at the beginning of CD34+ cells culture)and shall remain present, in the culture medium, for at least threedays, and preferably during all the time the cells are culture (i.e.preferably about 7 days).

When performing the method herein disclosed, it is possible to furtheradd StemRegenin 1 (SR1,4-(2-(2-(Benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol,CAS 1227633-49-10) in the culture medium already containing theTNF-alpha.

The invention also relates to a method for obtaining T cellsprogenitors, comprising the steps of

a. performing the method as disclosed above and

b. purifying the generated T cells progenitors thereby obtained.

Purification may be performed by washing the cells and resuspending suchin a basal medium.

This method may also comprise the step of conditioning the T cellsprogenitors in a pouch for injection to a patient.

In this case, it is preferred when these cells are reconditioned in asaline solution containing 5% HSA such as Albunorm™ 5% 50 g/L(Octopharma, Lingolsheim, France).

These cells may also be frozen according to methods known in the art.

The invention also relates to a pouch for intravenous injection,containing a population of T cells progenitors (susceptible to beobtained or as obtained by a method as described above), among which theproportion of CD7+ cells in this population is higher than 80%, morepreferably higher than 85%.

The invention also relates to a pouch for intravenous injection,containing a population of CD7+ T cells progenitors (susceptible to beobtained or as obtained by a method as described above), where theproportion of CD34- and CD5− cells (cells that are CD7+ and both CD34-and CD5-) in this population is higher than 80%, more preferably higherthan 85%.

The invention also relates to a pouch for intravenous injection,containing a population of CD7+ T cells progenitors (susceptible to beobtained or as obtained by a method as described above), where theproportion of CD34− cells (cells that are CD7+ and CD34-) in thispopulation is higher than 50%, more preferably higher than 60%.Furthermore, at least 80%, and more preferably at least 85% of the cellsin this population are CD1a− cells.

The invention also relates to an in vitro method for obtainingtransformed T-cells progenitors, comprising the steps of

-   -   a. culturing CD34+ cells in a suitable medium comprising        TNF-alpha and in the presence of an immobilized Notch ligand    -   b. exposing the cells to a vector intended for transfection or        transduction of CD34+ cells.

As disclosed above, and after steps of washing and culturing again thecells, transformed T-cells progenitors (expressing a transgene,integrated within their genome) are obtained.

The invention also relates to an in vitro method for obtaining modifiedT-cells progenitors, comprising the steps of

a. culturing CD34+ cells in a medium comprising TNF-alpha and in thepresence of an immobilized Notch ligand

b. exposing the cells to a vector or a nucleid acid sequence containingthe element appropriate for gene editing at least for some time duringcell culture.

Thereby T cells progenitors modified by gene editing are obtained, afterpotential steps of washing and culturing.

The invention also relates to T cells progenitors, in particular asobtained by the method herein disclosed, for their use in animmunosuppressed patient, in particular for allowing immunereconstitution in this patient and/or obtaining an immune protectionagainst infections in said patient, during a period of some months (atleast two months, preferably at least six months).

In a particular embodiment, the patient is an immunosuppressed patient.The reasons for the deficiency may be multiple: hereditary immunedeficiency, chemotherapy for leukemia, conditioning, graft containingonly stem cells, post-graft treatment for prophylaxis of GVH(graft-versus-host disease), age of patient, and complications such asinfections.

In particular, the patient can be immunosuppressed due to the depletionof its immune cells following therapy before hematopoietic stem cellstransplantation. In this embodiment, the graft may be an allograft (inthis case, the T cell progenitors are preferably derived from apartially HLA-compatible donor), or an autograft (in which case the Tcell progenitors have preferentially being transformed by a vector inorder to express a gene and/or a protein making it possible to correct agenetic defect in said patient).

The T cell progenitors are preferably a population of CD7+ cells (i.e. apopulation where at least 75%, more preferably at least 80% of cells inthe population express the CD7 marker). This population is also part ofthe invention. In particular, it is susceptible to be obtained by amethod herein disclosed. In a specific embodiment, it is obtained by amethod herein disclosed.

More specifically, the T cell progenitors are preferably a population ofCD7+ cells (i.e. a population where at least 75%, more preferably atleast 80% of cells in the population express the CD7 marker) where theproportion of CD34- and CD5-cells (cells that are CD7+ and both CD34-and CD5-) in this population is higher than 80%, more preferably higherthan 85%. This population is also part of the invention. In particular,it is susceptible to be obtained by a method herein disclosed. In aspecific embodiment, it is obtained by a method herein disclosed.

In another embodiment, the T cell progenitors are preferably apopulation of CD7+ cells (i.e. a population where at least 75%, morepreferably at least 80% of cells in the population express the CD7marker). In this population, more than 50%, more preferably more than60% of cells don't express the CD34 marker. In this population at least80%, more preferably at least 85% of cells don't express the CD1amarker. The proportion of CD7+CD1a− cells in this population ispreferably at least 80%. This population is also part of the invention.In particular, it is susceptible to be obtained by a method hereindisclosed. In a specific embodiment, it is obtained by a method hereindisclosed.

In order to determine whether a cell is positive or not to a surfacemarker (CD7, CD34, CD5 or CD1a), one shall use any method known in theart, and in particular flow cytometry, after the cells have been markedwith fluorescent antibodies directed against the surface antigen. Theprinciple is that a signal will be emitted, for each cell of thepopulation, with a given intensity, and cells are considered as positivefor the antigen if the signal is higher than a given threshold.

For CD34: a control population consisting of a population of HPSC (suchas one isolated from cord blood or from mobilized peripheral blood) isused to determine the appropriate thresholds. In this population ofcells, the cells are CD34+CD7−CD5−, or CD34+CD7−CD1a−. With thiscontrol, it is possible to determine threshold for each antigen thatwill be used to determine whether cells in another population arepositive or not for these antigens.

The control population generally will contain around 90% ofCD34+CD7−CD5−, or of CD34+CD7−CD1a− hematopoietic stem cells. Thethreshold is the signal intensity level for which the cells of thepopulation can be sorted in a 90/10 proportion.

For CD7 (resp. CD5 or CD1a): a control population is used, obtained byisolation of Peripheral Blood Mononuclear Cell (PBMC) by any techniqueknown in the art, and in particular a density gradient technique such asFicoll-Paque PLUS (GE Healthcare Life Sciences). CD7 (resp. CD5 or CD1a)positive cells are isolated using any technique known in the art such asthe MACS® technique (Magnetic Cell Isolation and Cell Separation,Miltenyi Biotec) which uses magnetic beads with anti-CD7 (resp. anti.CD5or anti-CD1a) antibody. In this population, the cells will essentiallybe CD7+(resp. CD5+ or CD1a+), it is believed that the proportion wouldbe around 90/10.

The thresholds for assessing whether a cell is positive to a surfaceantigen are determined using the control population for this antigen,and would be the intensity for which more than 90% of the cells of thecontrol population have a higher signal intensity.

Consequently, a cell is considered as being positive for an antigen ifthe intensity signal for this antigen is higher than the threshold asdetermined above, and negative for the antigen if the intensity signalfor this antigen is lower than the threshold as determined above.

The fact that the vast majority of T cell progenitors in the populationdon't express the CD1a marker may prove to be favorable, as cellsexpressing this CD1a marker may not be very efficient to reach thethymus and may thus be as efficient as CD1a− cells for in vivorepopulation. Since the reconstitution potential of CD1a+ cells isuncertain, it is thus preferable to lower the amount of such cells inthe population.

The invention also relates to a method for treating an immunosuppressedpatient, in particular for the purpose of allowing an immunereconstitution, at least temporarily, in this patient, comprising thestep of administering to said patient T-cell progenitors, as describedabove.

This method may also include the step of obtaining such progenitors byexposing CD34+ cells to a Notch ligand in the presence of TNF-alpha (asdescribed above) and preferably to a protein or peptide having the RGDSmotif and/or the CS1 motif, in particular A fibronectin fragment asdescribed above, under the conditions mentioned above.

In particular, a therapeutically effective amount is administered, thatis to say of the order of 1 to 5×10⁶ of progenitors per kg, which makesit possible to provide the patient with cells capable of playing aprotective role with regard to infections during a few months (of theorder of about 6 months).

Preferably, this administration of T cell progenitors is performed justprior to, just after or concomitantly with a hematopoietic stem celltransplant in said patient. As seen above, the injected cells can betransformed by a vector intended to allow the correction of a geneticdefect in said patient.

In another embodiment, the T cell progenitors are injected to a patientthat doesn't need a hematopoietic stem cell transplant. Indeed, it ispossible to use the transformed or transduced T cells progenitors totreat some immunodeficiencies, in which only T cells are affected, HIVor cancer patients (using the progenitors modified to lead to CAR-Tcells), without the need to perform the immune system depletivechemotherapy.

It is to be noted that the teachings of the invention, as disclosed withTNF-alpha are also applicable with the purine derivative StemRegenin 1(SR1, disclosed in Boitano et al, Science. 2010 Sep. 10;329(5997):1345-8). Thus SR1 can be substituted to TNF-alpha (i.e. usedin place of TNF-alpha) to obtain T cell progenitors from CD34+ cells,with a beginning of differentiation, and expansion of the cells(increase in the number of cells). The concentration of SR1 ispreferably in the range of 750 nM (30 ng/ml), also higher concentrationsuch as 1500 nM or 2500 nM (100 ng/ml), or even 5000 nM (200 ng/ml) orlower such as 500 nM (20 ng/ml) can also be envisaged. When used alone,but particularly in combination with TNF-alpha, much lowerconcentrations can be used, as low as 3 ng/ml, or 10 ng/ml. Suitableconcentrations also include 30 ng/ml or 100 ng/ml. Consequently, anyconcentration higher than 3 ng/ml, or higher than 10 ng/ml is suitableto increase the differentiation of the CD34+ cells to T-cellsprogenitors.

SR1 is a ligand (an antagonist) of the Aryl hydrocarbon/Dioxin receptor(AhR). Other AhR antagonists can be used alone, or in combination withTNF-alpha, to promote differentiation of CD34+ cells into the T-cellprogenitor lineage. One can cite resveratrol, omeprazole, Luteolin,alpha-naphthoflavone, mexiletine, tranilast, 6,2′,4′-Trimethoxyflavone,CH 223191(1-Methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide,CAS 301326-22-7). The amount of AhR antagonist is to be determined on acase-by-case basis by one of skill in the art, taking into considerationthe IC50 of the antagonist (SR1 has an IC50 of 127 nM, whereas CH 223191has an IC50 of 30 nM) and the fact that some products may have anagonist activity towards the AhR when used at high concentrations (suchas alpha-naphthoflavone) or depending of the cellular context (such asomeprazole). In view of the difference in IC50 between SR1 and CH223191, it is foreseen that an effective concentration of CH 223191would be well below the effective concentration of SR1, hereindisclosed.

Consequently, the invention also relates to

-   -   (i) an in vitro method for generating T cells precursors,        comprising the step of culturing CD34+ cells in a medium        comprising an antagonist of the Aryl hydrocarbon/Dioxin        receptor, in particular StemRegenin 1 (SR1) and in the presence        of an immobilized Notch ligand.    -   (ii) The in vitro method as above, wherein the culture medium        further contains TNF-alpha.    -   (iii) The in vitro method as above, wherein the antagonist of        the Aryl hydrocarbon/Dioxin receptor, in particular SR1, is        present, in the culture medium, from day 0 of the culture.    -   (iv) The in vitro method of any of above, wherein the CD34+        cells have been isolated from an adult donor, or from cord        blood.    -   (v) The in vitro method of any of above, wherein the cells are        cultured in presence of an antagonist of the Aryl        hydrocarbon/Dioxin receptor, in particular SR1, for at most 10        days.    -   (vi) The in vitro method of any of above, wherein the cells are        cultured in presence of the antagonist of the Aryl        hydrocarbon/Dioxin receptor, in particular SR1, between 3 and 7        days.    -   (vii) The in vitro method of any of above, wherein the        antagonist of the Aryl hydrocarbon/Dioxin receptor, in        particular SR1, is added to the medium culture at a        concentration between 1 ng/ml and 300 ng/ml and preferably        higher or equal to 1 ng/ml, or higher or equal to 3 ng/ml, or        higher or equal to 10 ng/ml, and preferably lower than 200        ng/ml, or 150 ng/ml and generally between 3 ng/ml and 100 ng/ml.    -   (viii) The in vitro method of any of above, wherein the Notch        ligand is the soluble domain of the Delta-like-4 ligand, fused        to a Fc region of an IgG protein, preferably an IgG2 protein.    -   (ix) The in vitro method of any of above, wherein the cells are        also exposed to a fibronectin fragment, wherein said fragment        comprises the RGDS and CS-1 patterns as well as a        heparin-binding domain, preferably immobilized on the inner        surface of the culture vessel.    -   (x) The in vitro method of above, wherein the fibronectin        fragment is Retronectin®, as disclosed above.    -   (xi) The in vitro method of any of above, wherein the culture        medium also contains a vector intended for transfection or        transduction of the CD34+ cells, during at least some time of        exposure of the CD34+ cells to the Notch ligand.    -   (xii) The in vitro method of any of above, wherein the culture        medium contains at least three, and preferably all four,        cytokines or growth factors chosen from the group consisting of        Interleukin 7 (IL-7), SCF (Stem Cells Factor), thrombopoietin        (TPO), and Flt3 ligand (FLT3L).    -   (xiii) An (in vitro) method for obtaining T cells progenitors,        comprising the steps of        -   a. performing the method according to any of above and        -   b. purifying the generated T cells progenitors        -   c. optionally conditioning the T cells progenitors in a            pouch for injection to a patient.    -   (xiv) An in vitro method for obtaining transformed T-cells        progenitors, comprising the steps of        -   a. culturing CD34+ cells in a medium comprising an            antagonist of the Aryl hydrocarbon/Dioxin receptor, in            particular SR1, and in the presence of an immobilized Notch            ligand        -   b. exposing the cells to a vector intended for transfection            or transduction of CD34+ cells.    -   (xv) An in vitro method for obtaining modified T-cells        progenitors, comprising the steps of        -   a. culturing CD34+ cells in a medium comprising an            antagonist of the Aryl hydrocarbon/Dioxin receptor, in            particular SR1, and in the presence of an immobilized Notch            ligand        -   b. exposing the cells to a vector or nucleic acid sequences            containing the element appropriate for gene editing.

The invention also relates to a kit for performing any method asindicated above, comprising:

-   -   (i) a coating medium containing a ligand of Notch (in particular        the soluble domain of the Delta-like-ligand, fused to a Fc        region of an IgG protein, in particular a IgG2 protein), and        optionally a fibronectin fragment    -   (ii) a medium adapted for culturing (and/or expanding) CD34+        cells and T cells such as α-MEM, DMEM, RPMI 1640, IMDM, BME,        McCoy's 5A, StemSpan™ SFII (StemCell Technologies) media,        X—VIVO™ medium or Fischer's medium    -   (iii) a progenitor expansion medium containing TNF-alpha and        preferably three cytokines, in particular selected from SCF,        TPO, Flt3L, and IL-7. It is preferred when the progenitor        expansion medium contains TNF-alpha and all four cytokines SCF,        TPO, Flt3L, and IL-7.

In another embodiment, the invention relates to a kit comprising thesame elements (i) and (ii) as indicated above, and a progenitorexpansion medium (iii) which contains an antagonist of the Arylhydrocarbon/Dioxin receptor, in particular SR1, and preferably threecytokines, in particular selected from SCF, TPO, Flt3L, and IL-7. It ispreferred when the progenitor expansion medium contains SR1 and all fourcytokines SCF, TPO, Flt3L, and IL-7.

In another embodiment, the progenitor expansion medium (iii) containsTNF-alpha and an antagonist of the Aryl hydrocarbon/Dioxin receptor, inparticular SR1, and preferably three cytokines, in particular selectedfrom SCF, TPO, Flt3L, and IL-7. It is preferred when the progenitorexpansion medium contains TNF-alpha, SR1 and all four cytokines SCF,TPO, Flt3L, and IL-7.

Such kit is particularly adapted and designed for performing the methodsherein disclosed.

The coating medium (i) is first used to coat the walls of a culturevessel.

The medium (ii) is then used to culture CD34+ cells (either obtainedfrom cord blood or from mobilized peripheral blood, in particular froman adult). Such medium is generally and preferably used at a 1×concentration (i.e. it can be used without dilution).

The medium (iii) is generally presented as a 10× dilution (i.e. it hasto be diluted in a medium (ii) for use. The reconstituted medium frommedia (ii) and (iii) is then used to promoter differentiation of theCD34+ cells to T-cell progenitors, in the T-cell lineage, as disclosedabove.

The invention also relates to a method for increasing the number of Tcells in a subject in need thereof, the method comprising administeringto the subject an effective number of progenitor T cells as obtained bya method disclosed herein.

The invention also relates to progenitor T cells as obtained by anymethod herein disclosed for their use for treating a subject in need ofan increased number of T cells.

In particular, the subject is a human.

In particular, the administered progenitor T cells are autologous. Inanother embodiment, the administered progenitor T cells are allogeneic.

In particular, the subject in need of the increased number of T cellshas a medical condition causing or resulting in lymphopenia, inparticular cancer, HIV infection, partial thymectomy, autoimmunedisease, and/or organ transplant.

DESCRIPTION OF THE FIGURES

FIG. 1: Total nucleated cells number obtained starting with 20000 CD34+cells from cord blood (CB, FIG. 1.A) or mobilized peripheral blood (mPB,FIG. 1.B), after 3 days (black bars) and 7 days (cumulative black andgrey bars) of culture. NC: not complemented; SR1: addition ofStemRegenin 1 (750 nM); TNF-alpha: addition of TNF-alpha (100 ng/ml);+(a), (b), (c): number of cells observed when the complements are addedfrom 0-3 days of culture (a), 0-7 days of culture (b) or 4-7 days ofculture (c).

FIG. 2: Number of CD7+ T-cell precursors obtained at day 7 starting with20000 CD34+ cells from cord blood (CB, FIG. 2.A) or mobilized peripheralblood (mPB, FIG. 2.B). Black bars: CD34+CD7+ cells; grey bars: CD34-CD7+cells. (a), (b), (c): number of cells observed when the complements areadded from 0-3 days of culture (a), 0-7 days of culture (b) or 4-7 daysof culture (c).

FIG. 3: Expression of BcI11b was analyzed on lived cells after 7 days ofculture obtained starting with CD34+ cells from cord blood (CB) ormobilized peripheral blood (mPB) cultured in the presence (grey bars) orabsence (black bars) of TNF-alpha.

FIG. 4: Combined effect of TNF-alpha and the Notch ligand DL4 on thenumber of CD7+ cells obtained at day 7 starting from CD34+ cord bloodcells (CB, black bars, left) or mobilized peripheral blood (mPB, greybars, right). (+/−means presence or absence of DL4 or TNF-alpha).

FIG. 5: Frequency of myeloid cells at day 7 obtained starting from CD34+cells from cord blood (CB) or mobilized peripheral blood (mPB) inpresence (grey bars) or not (black bars) of TNF-alpha (Mean±SEM).

FIG. 6: Total CD7+ cells number obtained from day 3 to day 7 in a doseresponse assay of TNF-alpha, starting with CD34+ cells from cord blood(CB, FIG. 6.A) or mobilized peripheral blood (mPB, FIG. 6.B).

FIG. 7: Proportion of CD34-CD7+ cells (grey bars) vs CD34+CD7+ cells(black bars) in a dose response assay of TNF-alpha starting with CD34+cells from cord blood (CB, FIG. 7.A) or mobilized peripheral blood (mPB,FIG. 7.B)

FIG. 8: Proportion of cells in the different phases of the cellularcycle. A. CB: cells differentiated from cord blood; B. cellsdifferentiated from mobilized peripheral blood cells. NC: noncomplemented; TNF-alpha: cultured in presence of TNF-alpha (20 ng/ml);SR1: cultures in presence of SR1 (30 ng/ml)

FIG. 9: percentage (A) and total number (B) of CD5+CD7+ cells culturedstarting with CD34+ cells from cord blood in presence of TNF-alphaand/or SR1 at various concentrations, after 7 days of culture.

EXAMPLES Example 1—Material and Methods Human Cells

Cord blood samples not eligible for banking were used for researchpurposes, following the provision of informed consent by the child'smother. Mobilized Peripheral Blood (mPB) samples were collected fromhealthy donors after G-CSF mobilization. Samples were directly enrichedfor CD34+ cells. The informed consent was given by each donor(Biotherapy Department, Necker Hospital, Paris).

Exposure of CD34+ Progenitor Cells to Notch Ligand DL-4

CD34+ cells from human CB or mobilized peripheral blood samples werecultured in 24-well plates or 6-well plates that had been coated withrecombinant human fibronection (RetroNectin®, Clontech/Takara) and DL-4(5 μg/ml, PX'Therapeutics, Grenoble, France). Coating was performed for2 h at 37° C., DL-4 coated wells were then blocked with bovine serumalbumin 2% (BSA) in phosphate-buffered saline (PBS) for 30 minutes at37° C. and washed with PBS. Cultures were initiated at a concentrationof 2×10⁴ cells/well or 1×10⁵ cells/well (for 24-well and 6-well platesrespectively) in α-MEM medium (Gibco, life Technology), supplementedwith NaHCO₃(7.5%) (Gibco, life Technology) and 20% defined fetal calfserum (Hyclone, Thermo Fisher Scientific, Illkirch, France) and therecombinant human cytokines interleukin-7 (IL-7), Flt3-ligand (Flt-3),stem cell factor (SCF) and thrombopoietin (TPO) (all at 100 ng/ml andall purchased from PeproTech Inc, Rocky Hill, N.J.) with or withoutTNF-α (R&D Systems, US). After 3 days of culture, the cells were halfreplaced by fresh medium. Cultured cells were analyzed byfluorescence-activated cell sorting (FACS) after 3 and 7 days of cultureon DL-4 respectively to exclude CD34-/CD7− myeloid cells from subsequentanalyses.

In Vitro T Cell Differentiation Assay on OP9/DL1 Cells

The T-lymphoid potential of native CD34+CB cells and TNF-α induced Tcell progenitors generated by exposure to DL-4 was assessed in OP9/DL-1co-cultures, as previously described (Six et al, Blood Cells Mol Dis.2011 Jun. 15; 47(1):72-8 and Six J Exp Med. 2007 Dec. 24;204(13):3085-93).

Quantitative, Real-Time Polymerase Chain Reactions Using RT2 ProfilerArray

CD7+ cells were sorted on Ariall after 7 days of culture. Total RNA ofsorted cell fractions from day 3 and day 7 was isolated with the RneasyMicro Kit (Qiagen, Courtaboeuf, France). RT2 Profiler PCR arrays wereperformed following the protocol detailed in the RT2 Profiler PCR ArrayHandbook (SA Biosciences, Frederick Md.).

Flow Cytometry Analysis and Cell Sorting

Monoclonal antibodies against human CD34 (AC136), CD3 (BW264/56), CD45(5B1) were purchased from Miltenyi Biotech (Bergisch Gladbach, Germany),and CD4 (SK4), CD7 (M-T701), CD25 (M-A251), 7-aminoactinomycin D (7AAD)were from BD Biosciences (San José, Calif.). Anti-human CD8 (RPAT8) wasfrom Sony Biotechnology (San Jose, USA). The Anti-human Ctip2 (BcI11b)antibody was from Abcam (Cambridge, UK).

Human cells were stained and analyzed using a Gallios analyzer (BeckmanCoulter, Krefeld, Germany). Cells from xenogenic recipients wereanalysed on a MACSQuant® apparatus (Miltenyi Biotech, Bergisch Gladbach,Germany). The data were analyzed using FlowJo software (Treestar,Ashland, Oreg.) after gating on viable, 7AAD-negative cells. Cellsubsets were sorted on an ARIA II system.

Cell Proliferation Assays

For cell proliferation assays, CD34+ cells from CB and mPB were labeledusing the CellTrace™ CFSE kit (Life Technologies, Carlsbad, Calif.)prior to culture with DL-4 and TNF-alpha (Life Technologies). The cells'staining intensity was measured prior to culture each day from day 3 today 7. CFSE-positive cell were analyzed on a Gallios cytometer (BeckmanCoulter).

Cell Cycle Assays

For cell cycle analysis, cells were stained with Hoechst33342 (Lifetechnology) and Ki67-PC5 (BD Bioscience) after fixed with Fixativereagent of PerFix-nc kit (Beckman Coulter) at room temperature for 15min, and added permeablizing reagent. The data were analyzed usingFlowJo software (version 10.2, Treestar, Ashland, Oreg.) after gating onviable, 7AAD-negative cells.

Adoptive Transfer of In Vitro-Generated T-Cell Progenitors Derived fromAdult HSPCs into NSG Neonates

All experiments and procedures with animals were performed in compliancewith the French Ministry of Agriculture's regulations on animalexperiments. The injection of in vitro generated human T-cellprogenitors in NSG mice has been approved by the Ministry of HigherEducation and Research (APAFIS 2101-2015090411495178v4).

The NSG (NOD-Scid(IL2Rg^(null))) mice (obtained from the JacksonLaboratory, Bar Harbor, Me., http://www.jax.org) were kept in apathogen-free facility. Progeny derived from mPB CD34+ HSPCs in 7-dayDL-4 cultures with or without TNF a (3×10⁵ or 1×10⁶) were injectedintra-hepatically into NSG neonates (0-4 days old). Control mice wereinjected with either 3×10⁵ non-cultured mPB CD34+ cells or 100 ul PBS.

Average engraftment levels of NSG mice were determined from 4 to 12weeks post-transplant. Flow cytometry analysis was performed on freshlycells collected from femur, thymus, peripheral blood and spleen. Cellswere treated with 1× red blood cell lysis buffer (Biolegend, US) andwashed before stained by antibodies.

Analysis of T Cell Receptor Diversity

TCR gene rearrangement analysis was performed in duplicate and on thetwo independently purified subsets (average is shown).

TCR-δ quantification (D δ2-D δ3, D δ2-J δ1, and D δ3-J δ1) was performedwith the listed sets of primers and probes.

The following were used for D δ2-D δ3 rearrangements:

D δ2, (SEQ ID No 9) 5′-CAAGGAAAGGGAAAAAGGAAGAA-3′; D δ3, (SEQ ID No 10)5′-TTGCCCCTGCAGTTTTTGTAC-3′; and D′3 probe, (SEQ ID No 11)5′-ATACGCACAGTGCTACAAAACCTACAGAGACCT-3′.

The following primers and probe were used for D δ2-J δ1 rearrangements:

D δ2, (SEQ ID No 12) 5′-AGCGGGTGGTGATGGCAAAGT-3′; J δ1, (SEQ ID No 13)5′-TTAGATGGAGGATGCCTTAACCTTA-3′; and J δ1 probe, (SEQ ID No 14)5′-CCCGTGTGACTGTGGAACCAAGTAAGTAACTC-3′

The following were used for D δ3-J δ1 rearrangements:

D δ3, (SEQ ID No 15) 5′-GACTTGGAGAAAACATCTGGTTCTG-3′;J δ1 primer and J δ1 probe.

The analysis of TCR rearrangements by multiplex fluorescent PCR wasperformed by separation of fluorochrome-labeled single stand PCRproducts in a capillary sequencing polymer and detected via automatedlaser scanning.

Apoptosis Assays

Cells were washed by cold PBS and resuspended in binding buffer at aconcentration of 1 million cells/ml. After adding 5 ul Annexin V-PE (BDBioscience) and 2 ul 7AAD, cells were incubated in the dark at roomtemperature for 15 min. Subsequently, cells were washed with 500 ulbinding buffer and resuspended in 100 ul binding buffer to be analyzedwithin 1 hour.

Example 2: Improvement on the Expansion and Differentiation of T-CellPrecursors

When CD34+ cells are cultured with DL-4, FIG. 1 shows that addition ofTNF-alpha to the cell culture medium makes it possible to multiply thetotal number of cells recovered at day 7 by 10 times as compared toculture without TNF-alpha, either when starting with CD34+ cells issuedfrom cord blood (FIG. 1.A) or from PB (FIG. 1.B).

FIG. 2 shows that addition of TNF-alpha to the cell culture medium makesit possible to multiply the number of CD7+ cells in by 20 to 40 times,either when starting with CD34+ cells issued from cord blood (FIG. 2.A)or from PB (FIG. 2.B). The improvement is especially high for theCD34-CD7+ cell population.

Example 3: Analysis of the Surface Markers of the T Cell Progenitors

The surface markers present at the surface of the cells obtained after 7days of culture were determined by flow cytometry.

Cord blood mPB CD34+ CD34− CD34+ CD34− CD34+ CD34− CD34+ CD34− CD7+ CD7+CD7− CD7− CD7+ CD7+ CD7− CD7− −TNFα 29.5 38.9 15.4 16.3 15.1 11.3 32.940.7 +TNFα 0.39 95.6 0.76 3.22 0.68 90.1 2.75 6.49

Cord blood mPB CD5+ CD5− CD5+ CD5− CD5+ CD5− CD5+ CD5− CD7+ CD7+ CD7−CD7− CD7+ CD7+ CD7− CD7− −TNFα 1.56 66.8 5.10⁻³ 31.6 0.069 26.4 0.02773.5 +TNFα 0.66 95.4 0.012 3.97 3.01 87.7 0.24 9.00

These tables show that addition of TNF-alpha leads to an increase in theproportion of CD7+ cells, without really increasing the proportion ofCD5+ cells.

After 7 day culture, HSPCs differentiate into CD34-CD7+CD5− T-cellprecursors.

The surface markers present at the surface of the cells obtained after10 days of culture were also determined by flow cytometry.

No expression of CD1a was found (data not shown).

The kinetics of modification of the surface markers was studied and itwas found that presence of TNF-alpha in the culture medium increases theproportion of CD7+ from day 4 up to day 7 (data not shown).

Example 4: Rearrangement of T Cell Receptors

DL-4 T-cell precursors do not exhibit any signs of TCR rearrangementwith or without TNF-alpha or SR1 after 7 days of culture (data notshown).

Specific analysis of rearrangement was performed:

Results of TCRdelta Rearrangements

Detection of Dδ2-Dδ3 rearrangements in CB-NC et CB-SR1. No otherTCRdelta rearrangements were detected. Results were in accordance withRQ-PCR quantification

Results of TCRgamma Rearrangements

No TCRgamma rearrangements were detected.

Results of TCRbeta rearrangements

No TCRbeta rearrangements were detected.

Example 5: T-Commitment of TNF a Induced T-Cell Precusors

BcI11b is an important transcriptional factor uniquely switched on sinceT-cell commitment and absolutely required for T-cell differentiation.

Intracellular staining on T-cell precursors cultured with TNF-alphashowed positive expression of BcI11b for both CD34+ cells issued fromcord blood, and mPB. FIG. 3 shows that TNF-alpha increases theproportion of total cells expressing BcI11b transcription factor. Whencultured with TNF-alpha, the proportion of BcI11b expressing cells wasincreased.

Example 6: Differentiation on Other Lineages

Presence of other cell surface markers (CD14 and CD33) specific of otherlineages was assessed.

FIG. 5 shows that the culture in the presence of TNF-alpha made suchcells not detectable, whereas their proportion is less than 22% whenCD34+ cells are cultured without TNF-alpha.

Example 7: TNF-Alpha Reduces Apoptosis of the Cells

Apoptosis markers (7AAD and Annexin5) were studied.

7AAD +/− AnnexinV + apoptotic cells (%) CB mPB −TNFα 1.66 10.56 +TNFα0.28 0.96

This table shows that culture in presence of TNF-alpha reduces thepresence of the apoptosis markers. This is particularly apparent formPB.

Example 8: Dose Response Assay of TNF-Alpha

Various doses of TNF-alpha were used.

FIG. 6 shows that TNFa may increase CD7+ cell numbers during culture,when the concentration is more than 10 ng/ml, either for CB cells (FIG.6.A) or mPB cells (FIG. 6.B).

Kinetics of the dose response showed that TNF-alpha increases the T-celldifferentiation after only 4 days of culture in DL-4. There was nodifference between the concentration 10, 50 and 100 ng/ml (not shown).

To determine the threshold of effective concentration, analysis of lowerconcentrations (0.01-10 ng/ml) was performed.

The effect of TNF-alpha on CB and mPB on T-cell differentiation(percentage of CD34− CD7+ cells) was found to be concentration-dependentat low concentration (FIG. 7). The total number of CD7+ T-cellprecursors was not different from 5 ng/ml to 100 ng/ml.

Example 9: Proliferation Analysis During Culture

TNF-alpha was found to increase the proliferation of CD34+CD7+ T-cellprecursors since day 3 in DL-4 culture as compared to culture conditionswithout TNF-alpha (data not shown).

Example 10: Synergy Between TNF-Alpha and the Notch Ligand

FIG. 4 shows that without DL4, both CB and mPB failed to differentiateinto CD7+ T-cell precursors. Even the complementation of the medium withTNF-alpha couldn't rescue it.

When both TNF-alpha and the Notch ligand are present, the effectobserved is very high. It thus seems that there is a synergy betweenthese two compounds and that the effect of TNF-alpha on T-celldifferentiation is likely Notch dependant.

Example 11: Addition of TNF-Alpha Increases Proliferation of CD7+Progenitors

After 7 days of culture in presence of TNF-alpha, CD34+CD7-, CD34+CD7+and CD34-CD7+ subsets were sorted, stained with CFSE (Carboxyfluoresceinsuccinimidyl ester) and the dilution of CFSE (surrogate marker of cellproliferation) was followed from day 8 to 10.

Only CD34+CD7+ and CD34-CD7+ cells show increased proliferation whencultured with TNF-alpha. (Data not shown)

Example 12: Cell Cycle Analysis

Analysis of the cell cycle was performed. It was observed that morecells were released from GO phase in presence of TNF-alpha on both CBand mPB derived CD7+ progenitors (FIG. 8).

Example 13: Combination of SR1 and TNFa

SR1 accelerates T-cell differentiation as shown by the presence ofCD5+CD7+ cells at day 7. The number of CD5+CD7+ cells is increased bythe presence of both TNF-alpha and SR1 (FIG. 9).

Example 14: In Vivo Data

T-cell precursors induced in presence of TNF-alpha may largely fastenthe reconstitution of the T-lineage in vivo.

Indeed, 4 weeks post-transplantation, recipient mice injected with mPBT-cell precursors produced in the presence of TNF-alpha have largerthymus than mice injected with mPB T-cell precursors produced withoutTNF-alpha. T-cell precursors induced in presence of TNF-alpha candifferentiate to activated TCRαβ T cells within 4 weeks in vivo. (Datanot shown)

In summary, addition of TNF-alpha from day 0 in the DL-4 culture systemleads to an increase of T-cell progenitors (defined by the surfaceexpression of CD7) of 40 fold for mPB HSPC and 20 fold for CB HSPC atday 7.

The CD7+ T-cell progenitors generated from both CB and mPB were mostlyCD34− and were CD1a negative. Cells were also mostly CD5 negative.

They expressed BcI11b, which is important fine-turning molecular forT-commitment and further T-cell differentiation.

They did not exhibit any signs of T-cell receptor rearrangements.

Their phenotype and molecular characteristics were similar to the one ofthe CD34-CD7+ T-cell progenitors obtained without TNF-alpha.

Regarding the mechanisms involved in TNF-alpha action, TNF-alphadecreases expression of apoptosis markers and increases cellproliferation during the culture. It also inhibits myeloid cellproduction.

The use of TNF-alpha in the DL4 culture system increase to a huge extentthe amounts of T-cell progenitors produced from both human adult andcord blood HSPC. It may thus overcome the difficulty to obtain largeamounts of T-cell progenitors from adult HSPC. It may also decrease thenumber of starting HSPC required in future clinical trials and thequantity of GMP grade and other reagents required, thus decreasing thecosts of production of these T-cell progenitors.

1.-20. (canceled)
 21. In vitro method for generating T cells precursors,comprising the step of culturing CD34+ cells in a medium comprisingTNF-alpha, and in the presence of an immobilized Notch ligand, whereinthe cells are also exposed to a fibronectin fragment, wherein saidfragment comprises the RGDS and CS-1 patterns as well as aheparin-binding domain.
 22. The in vitro method of claim 21, wherein theNotch ligand is immobilized on the inner surface of a culture vessel oron the surface of beads present in the culture medium.
 23. The in vitromethod of claim 21, wherein TNF-alpha is present, in the culture medium,from day 0 of the culture
 24. The in vitro method of claim 21, whereinTNF-alpha is present, in the culture medium, at a concentration higheror equal to 3 ng/ml.
 25. The in vitro method of claim 21, wherein theCD34+ cells have been isolated from an adult donor.
 26. The in vitromethod of claim 21, wherein the cells are cultured in presence ofTNF-alpha for at most 10 days.
 27. The in vitro method of claim 21,wherein the cells are cultured in presence of TNF-alpha between 3 and 7days.
 28. The in vitro method of claim 21, wherein the Notch ligand isthe soluble domain of the Delta-like-4 ligand, fused to a Fc region ofan IgG protein.
 29. The in vitro method of claim 21, wherein thefibronectin fragment is immobilized on the inner surface of the culturevessel or on beads.
 30. The in vitro method of claim 21, wherein thefibronectin fragment is Retronectin®.
 31. The in vitro method of claim21, wherein the culture medium also contains a vector intended fortransfection or transduction of the CD34+ cells, during at least sometime of exposure of the CD34+ cells to the Notch ligand.
 32. The invitro method of claim 31, wherein the vector intended for transfectionor transduction of the CD34+ cells is a transgene that codes for aChimeric Antigen Receptor (CAR).
 33. The in vitro method of claim 21,further comprising the step of a. purifying the generated T cellsprogenitors, and b optionally conditioning the T cells progenitors in apouch for injection to a patient.
 34. The in vitro method of claim 21,further comprising the step of exposing the cells to a vector intendedfor transfection or transduction of CD34+ cells.
 35. The in vitro methodof claim 21, further comprising the step of exposing the cells to avector or nucleic acid sequences containing the element appropriate forgene editing.
 36. A T cell progenitors population obtained by the methodof claim 21, wherein the proportion of cells that are CD7+ and CD34- andCD5− is higher than 60%.
 37. The T cell progenitors population accordingto claim 36, expressing a CAR.
 38. A method for increasing the number ofT cells in a subject in need thereof, comprising administering theprogenitor T cell according to claim 36 to a subject.
 39. The method ofclaim 38, for treating lymphopenia.
 40. The method of claim 38, fortreating cancer, HIV infection, partial thymectomy, autoimmune disease,and/or organ transplant.